GITRL on Inflammatory Antigen Presenting Cells in the Lung
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www.nature.com/mi ARTICLE GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T-cell accumulation and tissue-resident memory T-cell formation Kuan-Lun Chu1, Nathalia V. Batista1, Kuan Chung Wang1, Angela C. Zhou1 and Tania H. Watts1 T-cell responses in the lung are critical for protection against respiratory pathogens. TNFR superfamily members play important roles in providing survival signals to T cells during respiratory infections. However, whether these signals take place mainly during priming in the secondary lymphoid organs and/or in the peripheral tissues remains unknown. Here we show that under conditions of competition, GITR provides a T-cell intrinsic advantage to both CD4 and CD8 effector T cells in the lung tissue, as well as for the formation of CD4 and CD8 tissue-resident memory T cells during respiratory influenza infection in mice. In contrast, under non- competitive conditions, GITR has a preferential effect on CD8 over CD4 T cells. The nucleoprotein-specific CD8 T-cell response partially compensated for GITR deficiency by expansion of higher affinity T cells; whereas, the polymerase-specific response was less flexible and more GITR dependent. Following influenza infection, GITR is expressed on lung T cells and GITRL is preferentially expressed on lung monocyte-derived inflammatory antigen presenting cells. Accordingly, we show that GITR+/+ T cells in the lung parenchyma express more phosphorylated-ribosomal protein S6 than their GITR−/− counterparts. Thus, GITR signaling within the lung tissue critically regulates effector and tissue-resident memory T-cell accumulation. Mucosal Immunology (2019) 12:363–377; https://doi.org/10.1038/s41385-018-0105-5 INTRODUCTION checkpoint for CD4 accumulation and shows only indirect effects Influenza remains an important human pathogen. CD4 and CD8 on CD8 T cells.7,14 In contrast, in a T-cell adoptive transfer model of T cells play crucial roles in control of influenza virus.1,2 However, influenza infection, GITR was shown to mainly affect CD8 T-cell their activation must be tightly regulated in order to clear the responses, with critical effects on viral control.13 However, the role infection, avoid pathology, and at the same time allow the appro- of GITR in the endogenous T cell responses to influenza virus, and priate formation of memory T cells.3,4 The initial activation of when and where these signals take place are incompletely T cells requires an antigen bound to MHC (signal 1), a defined. Here, we provide evidence that GITRL on monocyte- costimulatory signal through CD28 (signal 2)5 as well as cytokines derived inflammatory APCs provides critical signals through GITR (signal 3).6 Although these signals are important in initiating T-cell on T cells in the mediastinal lymph node (mLN) and then again in responses, additional post-priming signals from tumor necrosis the lung tissue to allow effector T-cell accumulation during factor receptor (TNFR) superfamily members play critical roles in respiratory influenza infection. This signal is crucial for rescuing 7–12 b controlling the duration of the T-cell responses, referred to as low-affinity D /NP366–374-specific CD8 T cells as well as for optimal signal 4.7 formation of tissue-resident memory T cells (Trm) in the lung. Glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18, or CD357), a costimulatory member of the TNFR superfamily plays an important role in the control of viral infections, including RESULTS influenza virus and LCMV Clone 13.7,13–15 GITR is constitutively GITR is intrinsically required for effector CD4 and CD8 T-cell expressed at low levels on resting T cells and at high levels on accumulation during influenza infection CD4+ CD25+ regulatory T cells (Treg),16,17 and is rapidly To determine the intrinsic role of GITR on endogenous CD4 and upregulated on CD4 and CD8 effector T cells upon activation.16,17 CD8 T-cell responses during influenza infection, we generated GITR is also detected on other immune cell types such as B cells, mixed bone marrow chimeras in which Thy1.1 CD45.2 hosts were NK cells, and macrophages.18 GITR ligand (GITRL) is mainly lethally irradiated and reconstituted with bone marrow from expressed on antigen presenting cells (APCs) and endothelial Thy1.2 CD45.1 GITR+/+ and Thy1.2 CD45.2 GITR−/− mice mixed in cells,19 with monocyte-derived inflammatory APCs showing the a 1:1 ratio. This model allows us to assess the competition highest level of expression during chronic LCMV infection.7 between GITR+/+ and GITR−/− cells within the same mouse The role of GITR during viral infection appears context (Fig. 1a, b). Mice were allowed to reconstitute for 90 days and then dependent. In the chronic LCMV clone 13 infection model, GITR pre-infection ratios of donor CD45.1 and CD45.2 were determined is mainly required on CD4 T cells to provide a post-priming in the blood. In another set of bone marrow chimeras, we 1Department of Immunology, University of Toronto, Toronto, ON, Canada Correspondence: Tania H. Watts ([email protected]) Received: 19 March 2018 Revised: 2 October 2018 Accepted: 27 October 2018 Published online: 28 November 2018 © Society for Mucosal Immunology 2018 GITRL on inflammatory antigen presenting cells in the lung parenchyma. K-L Chu et al. 364 1234567890();,: analyzed the reconstitution ratio of GITR+/+ to GITR−/− CD4 or X31, GITR+/+ Th1 cells in the reconstituted mice showed a CD8 T cells in the spleen and mLN and found that these ratios competitive advantage over GITR−/− Th1 cells in the lung, mLN were indistinguishable from the ratio measured in the blood (data and spleen, with a more dramatic effect in the lung (Fig. 1c, d). not shown). Therefore, for each mouse, normalization was done These effects were greater at the peak of the response, day 10 using the pre-infection ratio of GITR+/+ to GITR−/− T cells post infection (p.i.), compared to day 7 p.i. GITR+/+ Tregs had a measured in the blood. Following infection with influenza A/HK- 1.5- to 2-fold advantage over GITR−/− Tregs in the three organs Mucosal Immunology (2019) 12:363 – 377 GITRL on inflammatory antigen presenting cells in the lung parenchyma. K-L Chu et al. 365 Fig. 1 Under conditions of competition, GITR is intrinsically required for the accumulation of effector CD4 and CD8 T cells during influenza infection. a Schematic indicating that lethally irradiated Thy1.1 CD45.2 hosts were reconstituted with a 1:1 mixture of GITR−/− Thy1.2 CD45.2: GITR+/+ Thy1.2 CD45.1 bone marrow cells. Chimeric mice were rested for 90 days and chimerism checked in the blood of each mouse, before intranasal (i.n.) infection with influenza A/HK-X31 followed by analysis at day 7 or day 10 post infection (p.i.). b Representative gating strategy for donor CD44hi T-bet+ Th1, CD25hi FoxP3+ Treg, and tetramer+ CD8 T cells. c Representative flow cytometry plots showing proportions of GITR+/+: GITR−/− of blood CD4 T cells before infection and of lung Th1 day 10 p.i. The normalized GITR+/+: GITR−/− ratio in Th1 d and Treg e compartments was evaluated in the lung, mediastinal lymph node (mLN), and spleen day 7 or day 10 p.i. Normalization was done by dividing the post-infection ratio in each mouse in each tissue by the pre-infection ratio of CD4 T cells in blood from the same mouse. f The normalized GITR+/+: GITR−/− ratio in CXCR5+ PD-1+ FoxP3−Tfh compartment was evaluated in the mLN and spleen day 7 p.i. and normalization was done as described above. Representative gating for donor Tfh is shown on the left. g Representative flow cytometry plots showing proportions of +/+ −/− b +/+ −/− GITR :GITR of blood CD8 T cells before infection and of lung D /PA224–233-specific CD8 T cells day 10 p.i. The normalized GITR :GITR b b ratio in D /NP366–374-specific CD8 T cell h and D /PA224–233-specific CD8 T cell i compartments was evaluated in the lung, mLN, and spleen day 7 or day 10 p.i. Normalization was done using pre-infection ratio of blood CD8 T cells as described for CD4 T cells above. Each symbol represents an individual mouse, with bars indicating median with interquartile range (IQR). Statistical analyses performed using Wilcoxon test comparing pre- and post-infection ratios. Data are pooled from 11 to 16 individual chimeric mice from at least two independent experiments Fig. 2 GITR is intrinsically required for the accumulation of IFNγ-producing CD4 and CD8 T cells during influenza infection. Mixed bone marrow chimeras were generated and infected as described in Fig. 1. Freshly isolated cells from the lung and splenocytes from chimeric mice b were restimulated with live influenza virus for 18 h a, b or with D -restricted influenza peptides: NP366–374 or PA224–233 for 6 h c, d, after which intracellular cytokines were measured by flow cytometry. a Representative flow cytometry plots depicting CD4+IFNγ+ T cells in the GITR+/+ and GITR−/− compartments day 7 p.i. in the lung. The normalized GITR+/+: GITR−/− ratio in CD4+IFNγ+ T cell b, CD8+IFNγ+ T cell (NP restim) c, and CD8+IFNγ+ T cell (PA restim) d compartments was evaluated at day 7 or day 10 p.i. in the lung and spleen. Normalization was done as described in Fig. 1. Each symbol represents an individual mouse, with bars indicating median with IQR. Statistical analyses performed using Wilcoxon test comparing pre- and post-infection ratios. Data were pooled from 10 to 16 individual chimeric mice from at least two independent experiments analyzed at day 7 p.i.